Project Summary The neurotransmitter dopamine (DA) plays a critical role in movement, reward, attention, and executive function. Several critically important neuropsychiatric disorders are associated with dysfunction in DA signaling including schizophrenia, obsessive compulsive disorder, bipolar disorder, autism, and attention deficit hyperactivity disorder (ADHD). Attention-Deficit Hyperactivity Disorder (ADHD) is a prevalent psychiatric disorder diagnosed solely on the basis of behavioral features. Although the biological underpinnings of ADHD remain ill-defined, evidence from basic, pharmacological and genetic studies have drawn attention to potential contributions of altered expression/function of the DA transporter (DAT). Thus, DA signaling modulates key elements of the behaviors altered in ADHD, psychostimulants that target DAT, including methylphenidate (MPH, Ritalin) and various amphetamine (AMPH) formulations (e.g. Adderall), rapidly relieve ADHD symptoms in many subjects, and human genetic studies reveal increased ADHD risk associated with DAT gene (SLC6A4) polymorphisms. Our lab identified multiple rare DAT coding variants in ADHD probands, one of which, DAT Val559, is the subject of the current proposal. Interestingly, DAT Val559 has not only been identified in ADHD subjects, but also in bipolar disorder and autism, suggesting contributions to DA-dependent traits that can manifest as risk in distinct disorders or contribute to ADHD comorbidities therein. Through in vitro studies, we demonstrated that the Val559 variant produces anomalous DA efflux (ADE), a form of non-vesicular DA release, typically seen with AMPH treatment of DAT-expressing cells. In the case of DAT Val559, AMPH fails to trigger DA efflux, though both AMPH and MPH block ADE. These findings lead to the hypothesis that tonic DA leak may be an unsuspected contributor to risk for one or more DA-linked behavioral disorders. To test this concept in vivo, we developed DAT Val559 knock-in mice that expresses the variant from the native Slc6a3 locus, affording the first construct valid model of DAT dysfunction associated with neuropsychiatric disorders. Here I propose to test the hypotheses that 1) in vivo expression of DAT Val559 generates altered neurotransmitter release as well as changes in pre and post-synaptic signaling events that are sensitive to ADHD-relevant pharmacological manipulations and 2) that lifelong DAT Val559 expression leads to alterations in DA-associated behaviors that align with one or more attributes of ADHD. Altogether, my efforts are organized to afford a rich training opportunity in the investigation of mechanisms that underlie risk for neuropsychiatric disorders while advancing analysis of the first construct-valid model of DA dysfunction in ADHD.